Recently, receptors of humoral factors having important physiological activities, typified by cytokine and hormones, have been revealed by gene manipulation, and a large number of receptors have been isolated and identified. The examples include receptors of lipophilic hormones generally referred to as intracellular receptors. As a result of the analysis of amino acid sequences of various intracellular receptors, it was revealed that they are ligand-dependent transcription factors having a common basic structure and forming one of gene family. That is, they belong to a family including a steroid receptor, such as glucocorticoid receptor, progesterone receptor, mineral corticoid receptor, androgen receptor, estrogen receptor and the like; a retinoid receptors, such as retinoid X receptor, retinoic acid receptor and the like; a peroxisome proliferator-activated receptor; a vitamin D3 receptor; and a thyroid hormone receptor. Since it has been revealed that functional regions, such as a ligand binding domain and a domain which recognizes a target DNA sequence and the like, are distinctively separated in these receptors (see Science, 240, 889 (1988)), they have been isolated based on the homology, though there are certain receptors whose ligands are still unknown.
Recently, a nuclear receptor, peroxisome proliferator-activated receptor (hereinafter referred to as a “PPAR receptor”), is drawing attention in the field of studies on transcription factors related to the expression and induction of fat cell differentiation marker genes. Regarding the PPAR receptor, cDNA has been cloned from various animal species, and a plurality of isoform genes have been found including α, δ and γ types in mammals. In addition, R1 is known that the γ0 type is expressed in fat tissues, immunocytes, adrenal gland, spleen and small intestine, and the a type in liver and retinas, but the δ type has no tissue specificity and is expressed ubiquitously.
On the other hand, Fas antigen is a membrane protein whose participation in programmed cell death, namely apoptosis has been clearly shown. Yonehara et al. have prepared monoclonal antibodies for human cell surface antigens and obtained an anti-Fas antibody showing lethal activity upon various human cells (see J. Exp. Med., 169, 1747 (1989)), cDNA of a cell surface molecule recognizable by the anti-Fas antibody has been isolated, and structure of the human Fas antigen has been determined (SEQ ID NO:22) (see Cell, 66, 233 (1991)). This Fas antigen is composed of 335 amino acid residues, and 16 amino acid residues in the N-terminus is assumed to be its signal peptide. A transmembrane region composed of 17 hydrophobic amino acid residues is present in a central position of the molecule, and it is considered that 157 amino acid residues in the N-terminus exist as its extracellular region and the C-terminal side sequence of 145 amino acid residues is its cytoplasmic region.
Since the structure of the Fas antigen is resemble in the structure of a receptor for tumor necrosis factor (TNF), it is assumed that the apoptosis of the Fas antigen occurs by a mechanism similar to the action of TNF. Functional regions of the Fas antigen have also been revealed gradually, and it has been found that the region essential for the signal transduction of apoptosis (functional region) is an amino acid sequence of the 175 h to 304′ positions (see J. Biol. Chem, 268, 10932 (1993)). In addition, the amino acid sequence of mouse Fas antigen has also been revealed (SEQ ID NO:23) (see J. Immunology, 148, 1274 (1992)), and it has a homology of 49.3% to the human Fas antigen as a whole. It is considered also that its functional region is an amino acid sequence of the 166th to 291st positions corresponding to the region of the human Fas antigen.
Receptors have a ligand binding region and a signalling transduction region. When a ligand is linked to the ligand binding region, the stereostructure of the signal transfer region is changed and a signal is transferred to other protein or DNA.
Recently, many studies have been conducted positively on the intercellular signal transduction by a fusion protein in which the ligand binding region of a receptor is linked to the signalling region of a different protein.
For example, it has been revealed that, in the case of cells transformed with a gene encoding a fusion protein of the ligand binding region of an estrogen receptor which is one of steroid receptors family, and the fanctional region of human cancer gene c-Myc, the cells are cancerated to abnormally proliferate by the stimulation of the ligand, estrogen (see Nature, 340, 66 (1989)).
Similar studies have been carried out on fusion proteins of the ligand binding region of each of the receptors for glucocorticoid, mineral corticoid and estrogen with the functional region of each of the proteins of E1A (adenovirus), c-Fos, v-Myb, C/EBP, v-Re1, GATA-1, 2 and 3, GAL4-VP16, Rev (HIV) and c-Ab1 (see Cell, 54, 1073 (1988), Proc. Natl. Acad. Sci. USA, 88, 5114 (1991), EMBO J., 10, 3713 (1991), Science, 251, 288 (1991), EMBO J., 11, 4641 (1992), Genes Dev. (in press), Proc. Nat. Acad. Sci. USA, 90, 1657 (1993), ibid., 87, 7787 (1990) and EMBO J., 12, 2809 (1993)), confirming that respective signals are transferred by the binding of ligands to their corresponding nuclear receptors.
Kakizuka et al. have reported a method in which the Fas antigen is used as a signal transduction protein for applying the above fusion protein to the treatment of a cancer (see JP-A-7-316200). In this method, an amino acid sequence of the 136th to 305th positions including not only the functional region but also the transmembrane region is fused with the ligand binding region of the intracellular receptor. As a result, a ligand capable of interacting with the amino acid sequence of the nuclear receptor ligand binding region can be detected effectively.
On the other hand, R. M. Evans et al. have reported a reporter assay method in which a reporter using an enzyme capable of converting a substrate into a chemiluminescent or visible dye product or introducing a substituent (e.g., luciferase, β-galactosidase, secretory alkaline phosphatase, or chloramphenicol acetyltransferase) in a region downstream of the Gal4 responsive element and a protein prepared by fusing the Gal4 DNA binding domain and the PPAR receptor ligand binding region are introduced into cells (see PCT International Application International Publication No. 9640128). Another assay method has also been reported in which, either by expressing an exogenous PPAR receptor protein or along with an endogenous PPAR receptor protein, the above reporter, with the proviso that in this case, the reporter is a reporter using the above enzyme in a region downstream of PPAR receptor responsive element (hereinafter referred to as “PPRE”) are introduced (Cell, 0, 803 (1995)). These assay systems are detection systems which can evaluate the function of intracellular receptors as transcription factors.
However, since these methods require a certain period of time for the activity detection, it is difficult to carry out high speed screening of the effect of ligands and compounds to be tested. In addition, since the introduction of DNA into cells is transient, it causes a problem in that the results cannot easily be compared due to insufficient stability among tests.